Methionyl tRNA synthetase polynucleotides

ABSTRACT

The invention provides metS polypeptides and DNA (RNA) encoding metS polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing metS polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims benefit of UK application number 9607999.1,filed Apr. 18, 1996.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the methionyl tRNA synthetase family, hereinafterreferred to as "metS".

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This has created a demandfor both new anti-microbial agents and diagnostic tests for thisorganism.

The t-RNA synthetases have a primary role in protein synthesis accordingto the following scheme: ##STR1## in which AA is an amino acid.

Inhibition of this process leads to a reduction in the levels of chargedt-RNA and this triggers a cascade of responses known as the stringentresponse, the result of which is the induction of a state of dormancy inthe organism. As such selective inhibitors of bacterial t-RNA synthetasehave potential as antibacterial agents. One example of such is mupirocinwhich is a selective inhibitor of isoleucyl t-RNA synthetase. Othert-RNA synthetases are now being examined as possible anti-bacterialtargets, this process being greatly assisted by the isolation of thesynthetase.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown Bacillus stearothermophilus methionyl tRNA synthetase protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel metS polypeptides by homology between the amino acidsequence set out in Table 1 [SEQ ID NO: 2] and a known amino acidsequence or sequences of other proteins such as Bacillusstearothertophilus methionyl tRNA synthetase protein.

It is a further object of the invention to provide polynucleotides thatencode metS polypeptides, particularly polynucleotides that encode thepolypeptide herein designated metS.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding metS polypeptides comprisingthe sequence set out in Table 1 [SEQ ID NO: 1] which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel metS protein from Streptococcus pneumoniae comprising the aminoacid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Streptococcus pneumoniae 0100993 strain contained in thedeposited strain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding metS, particularly Streptococcus pneumoniaemetS, including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of metS and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStreptococcus pneumoniae referred to herein as metS as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of metS polypeptide encoded by naturally occurring alleles ofthe metS gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned metS polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing metSexpression, treating disease, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid, assaying genetic variation, andadministering a metS polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStreptococcus pneumoniae bacteria In accordance with certain preferredembodiments of this and other aspects of the invention there areprovided polynucleotides that hybridize to metS polynucleotidesequences, particularly under stringent conditions.

In certain preferred embodiments of the invention there are providedantibodies against metS polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided metS agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a metS polynucleotide or a metS polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, N.Y., 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, N.Y., 1993; ComputerAnalysis of Sequence Data, Part I, Griffm, A. M., and Griffin, H. G.,eds., Humana Press, N.J., 1994; Sequence Analysis in Molecular Biology,von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, N.Y., 1991; andCarillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% "identity" to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously , by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO: 2 is intended that the amino acid sequenceof the polypeptide is identical to the reference sequence except thatthe polypeptide sequence may include up to five amino acid alterationsper each 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, N.Y. (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, N.Y. (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel metS polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel metS of Streptococcuspneumoniae, which is related by amino acid sequence homology to Bacillusstearothermophilus methionyl tRNA synthetase polypeptide. The inventionrelates especially to metS having the nucleotide and amino acidsequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2]respectively, and to the metS nucleotide sequences of the DNA in thedeposited strain and amino acid sequences encoded thereby.

                                      TABLE 1                                     __________________________________________________________________________    metS Polynucleotide and Polypeptide Sequences                                 __________________________________________________________________________    (A) Sequences from Streptococcus pneumoniae metS polynucleotide sequence      [EQ ID NO:1]                                                                    5'-1 ATGTCTGAAA AAAATTTTTA TATTACGACG CCGATTTACT ATCCATCTGG                    -   51 GAAACTTCAT ATCGGTTCTG CCTACACAAC TATCGCATGT GATGTCCTAG                 -  101 CACGTTACAA ACGCCTGATG GGNTACGATG TATTTTATCT GACAGGTCTT                 -  151 GATGAACATG GTCAGAAAAT CCAGCAGAAA GCGGAAGAAG CTGGTATTAC                 -  201 ACCTCAAGCC TATGTTGATG GAATGGCGGT TGGAGTTAAA GAACTCTGGC                 -  251 AATTACTAGA TATCTCATAC GATAAATTTA TCCGTACAAC CGATGACTAC                 -  301 CATGAAAAAG TTGTCGCACA GGTCTTTGAA CGCTTACTTG CTCAAGATGA                 -  351 TATCTACTTG GGTGAATACT CTGGTTGGTA TTCAGTATCA GACGAAGAAT                 -  401 TCTTTACAGA AAGCCAGCTG GCAGAAGTTT TCCGTGATGA AGCTGGAAAT                 -  451 GTGACTGGTG GTATTGCTCC ATCAGGTCAT GAGGTTGAAT GGGTTTCTGA                 -  501 AGAATCATAC TTCCTTCGCC TTAGCAAATA CCAAGACCGT TTGGTCGAAT                 -  551 TTTTCAAAGC TCATCCTGAA TTTATCACGC CAGATGGTCG TCTTAATGAA                 -  601 ATGCTACGCA ACTTCATCGA GCCAGGTTTG GAAGATTTGG CGGTATCTCG                 -  651 TACAACCTTT ACATGGGGAG TGCCTGTCCC ATCAAATCCA AAACACGTTG                 -  701 TCTACGTTTG GATTGATGCC CTTCTTAACT ATGCGACAGC TCTTGGCTAC                 -  751 GCTCAAGACG AACATGGTAA CTTTGACAAG TTCTGGAATG GAACAGTCTT                 -  801 CCATATGGTA GGAAAAGACA TCCTTCGCTT CCACTCTATC TACTGGCCAA                 -  851 TCCTTCTTAT GATGTTGGAT GTTAAATTAC CTGATCGTTT GATTGCCCAT                 -  901 GGTTGGTTTG TCATGAAAGA CGGAAAAATG TCTAAGTCAA AAGGGAATGT                 -  951 CGTTTACCCT GAAATGTTGG TAGAGCGTTA TGGACTAGAT CCACTTCGTT                 - 1001 ACTACCTCAT GCGTAACCTT CCAGTTGGTT CAGACGGAAC CTTTACTCCT                 - 1051 GAAGACTATG TCGGTCGTAT CAACTATGAA TTGGCTAATG ACCTTGGGAA                 - 1101 CCTCCTTAAC CGTACGGTTT CCATGATTAA TAAGTACTTT GATGGACAAA                 - 1151 TCCCTGCCTA TGTAGAAGGT GTGACTGAAT TTGATCATGT TCTTGCTGAG                 - 1201 GTTGCAGAAA AATCAATCGC AGACTTCCAT ACACACATGG AAGCAGTTGA                 - 1251 TTATCCACGT GCGCTTGAAG CAGTCTGGAC TCTGATCTCT CGTACCAATA                 - 1301 AATACATCGA TGAGACTGCA CCATGGGTCT TGGACAAGGA TGAAGCTCTT                 - 1351 CGTGACCAAT TGGCAAGTGT CATGAGCCAT TGGCAAGCCA GCATTCGTGT                 - 1401 AGTTGCTCAC TTGATTGAAC CATTTATGAT GGAAACTAGT CGTGCAGTTT                 - 1451 TGACTCAAAT TGGTTTGGAA GAAGTTTCTA GTCTTGAAAA CTTGAGTTTG                 - 1501 GCTGACTTCC CAGCAGATGT GACTGTAGTT GCCAAAGGAA CACCTATCTT                 - 1551 TCCACGTCTA AATATGGAAG AAGAAATCGC CTATATCAAG GAACAAATGG                 - 1601 AAGGCAATAA ACCAGCAGTC GAAAAAGAAT GGAATCCGGA CGAAGTTGAG                 - 1651 CTCAAACTAA ACAAGGATGA AATCAAGTTT GAAGACTTTG ACAAGGTTGA                 - 1701 AATCCGTGTC GCAGAAGTCA AAGAAGTGTC TAAAGTAGAA GGTTCAGATA                 - 1751 AGTTGCTTCA ATTCCGCTTG GATGCTGGTG ATGGAGAAGA TCGTCAGATT                 - 1801 CTTTCAGGAA TTGCAAAATA CTATCCAAAT GAACAAGAAT TGGTCGGCAA                 - 1851 GAAAGTTCAA ATCGTTGCTA ACCTCAAACC ACGTAAAATG ATGAAAAAAT                 - 1901 ATGTCAGCCA GGGTATGATT CTCTCAGCTG AACATGATGG CAAATTAACC                 - 1951 CTTCTCACAG TTGATCCAGC TGTACCAAAT GGAAGTGTGA TTGGGTAA-3'               - (B)  metS polypeptide sequence deduced from the polynucleotide            sequence in this table                                                          [SEQ ID NO:2].                                                                NH.sub.2 -1 MSEKNFYITT PIYYPSGKLH IGSAYTTIAC DVLARYKRLM GYDVFYLTGL            -    51 DEHGQKIQQK AEEAGTTPQA YVDGMAVGVK ELWQLLDISY DKFIRTTDDY                -   101 HEKVVAQVFE RLLAQDDIYL GEYSGWYSVS DEEFFTESQL AEVFRDEAGN                -   151 VTGGIAPSGH EVEWVSEESY FLRLSKYQDR LVEFFKAHPE FITPDGRLNE                -   201 MLRNFIEPGL EDLAVSRTTF TWGVPVPSNP KHVVYVWIDA LLNYATALGY                -   251 AQDEHGNFDK FWNGTVFHMV GKDILRFHSI YWPILLMMLD VKLPDRLTAH                -   301 GWFVMKDGKM SKSKGNVVYP EMLVERYGLD PLRYYLMRNL PVGSDGTFTP                -   351 EDYVGRINYE LANDLGNLLN RTVSMINKYF DGQIPAYVEG VTEFDHVLAE                -   401 VAEKSIADFH THMEAVDYPR ALEAVWTLIS RTNKYIDETA PWVLDKDEAL                -   451 RDQLASVMSH WQASIRVVAH LIEPFMMETS RAVLTQIGLE EVSSLENLSL                -   501 ADFPADVTVV AKGTPIFPRL NMEEEIAYIK EQMEGNKPAV EKEWNPDEVE                -   551 LKLNKDEIKF EDFDKVEIRV AEVKEVSKVE GSDKLLQFRL DAGDGEDRQI                -   601 LSGIAKYYPN EQELVGKKVQ IVANLKPRKM MKKYVSQGMI LSAEHDGKLT                -   651 LLTVDPAVPN GSVIG-COOH                                                 - (C) Polynucleotide sequence embodiments [SEQ ID NO:1].                     X-(R.sub.1).sub.n -1 ATGTCTGAAA AAAATTTTTA TATTACGACG CCGATTTACT             ATCCATCTGG                                                                       -        51 GAAACTTCAT ATCGGTTCTG CCTACACAAC TATCGCATGT GATGTCCTAG           -       101 CACGTTACAA ACGCCTGATG GGNTACGATG TATTTTATCT GACAGGTCTT            -       151 GATGAACATG GTCAGAAAAT CCAGCAGAAA GCGGAAGAAG CTGGTATTAC            -       201 ACCTCAAGCC TATGTTGATG GAATGGCGGT TGGAGTTAAA GAACTCTGGC            -       251 AATTACTAGA TATCTCATAC GATAAATTTA TCCGTACAAC CGATGACTAC            -       301 CATGAAAAAG TTGTCGCACA GGTCTTTGAA CGCTTACTTG CTCAAGATGA            -       351 TATCTACTTG GGTGAATACT CTGGTTGGTA TTCAGTATCA GACGAAGAAT            -       401 TCTTTACAGA AAGCCAGCTG GCAGAAGTTT TCCGTGATGA AGCTGGAAAT            -       451 GTGACTGGTG GTATTGCTCC ATCAGGTCAT GAGGTTGAAT GGGTTTCTGA            -       501 AGAATCATAC TTCCTTCGCC TTAGCAAATA CCAAGACCGT TTGGTCGAAT            -       551 TTTTCAAAGC TCATCCTGAA TTTATCACGC CAGATGGTCG TCTTAATGAA            -       601 ATGCTACGCA ACTTCATCGA GCCAGGTTTG GAAGATTTGG CGGTATCTCG            -       651 TACAACCTTT ACATGGGGAG TGCCTGTCCC ATCAAATCCA AAACACGTTG            -       701 TCTACGTTTG GATTGATGCC CTTCTTAACT ATGCGACAGC TCTTGGCTAC            -       751 GCTCAAGACG AACATGGTAA CTTTGACAAG TTCTGGAATG GAACAGTCTT            -       801 CCATATGGTA GGAAAAGACA TCCTTCGCTT CCACTCTATC TACTGGCCAA            -       851 TCCTTCTTAT GATGTTGGAT GTTAAATTAC CTGATCGTTT GATTGCCCAT            -       901 GGTTGGTTTG TCATGAAAGA CGGAAAAATG TCTAAGTCAA AAGGGAATGT            -       951 CGTTTACCCT GAAATGTTGG TAGAGCGTTA TGGACTAGAT CCACTTCGTT            -      1001 ACTACCTCAT GCGTAACCTT CCAGTTGGTT CAGACGGAAC CTTTACTCCT            -      1051 GAAGACTATG TCGGTCGTAT CAACTATGAA TTGGCTAATG ACCTTGGGAA            -      1101 CCTCCTTAAC CGTACGGTTT CCATGATTAA TAAGTACTTT GATGGACAAA            -      1151 TCCCTGCCTA TGTAGAAGGT GTGACTGAAT TTGATCATGT TCTTGCTGAG            -      1201 GTTGCAGAAA AATCAATCGC AGACTTCCAT ACACACATGG AAGCAGTTGA            -      1251 TTATCCACGT GCGCTTGAAG CAGTCTGGAC TCTGATCTCT CGTACCAATA            -      1301 AATACATCGA TGAGACTGCA CCATGGGTCT TGGACAAGGA TGAAGCTCTT            -      1351 CGTGACCAAT TGGCAAGTGT CATGAGCCAT TGGCAAGCCA GCATTCGTGT            -      1401 AGTTGCTCAC TTGATTGAAC CATTTATGAT GGAAACTAGT CGTGCAGTTT            -      1451 TGACTCAAAT TGGTTTGGAA GAAGTTTCTA GTCTTGAAAA CTTGAGTTTG            -      1501 GCTGACTTCC CAGCAGATGT GACTGTAGTT GCCAAAGGAA CACCTATCTT            -      1551 TCCACGTCTA AATATGGAAG AAGAAATCGC CTATATCAAG GAACAAATGG            -      1601 AAGGCAATAA ACCAGCAGTC GAAAAAGAAT GGAATCCGGA CGAAGTTGAG            -      1651 CTCAAACTAA ACAAGGATGA AATCAAGTTT GAAGACTTTG ACAAGGTTGA            -      1701 AATCCGTGTC GCAGAAGTCA AAGAAGTGTC TAAAGTAGAA GGTTCAGATA            -      1751 AGTTGCTTCA ATTCCGCTTG GATGCTGGTG ATGGAGAAGA TCGTCAGATT            -      1801 CTTTCAGGAA TTGCAAAATA CTATCCAAAT GAACAAGAAT TGGTCGGCAA            -      1851 GAAAGTTCAA ATCGTTGCTA ACCTCAAACC ACGTAAAATG ATGAAAAAAT            -      1901 ATGTCAGCCA GGGTATGATT CTCTCAGCTG AACATGATGG CAAATTAACC            -      1951 CTTCTCACAG TTGATCCAGC TGTACCAAAT GGAAGTGTGA TTGGGTAA              - -(R.sub.2).sub.n -Y                                                         - (D) Polypeptide sequence embodiments [SEQ ID NO:2].                        X-(R.sub.1).sub.n -1 MSEKNFYITT PIYYPSGKLH IGSAYTTIAC DVLARYKRLM             GYDVFYLTGL                                                                       -        51 DEHGQKIQQK AEEAGITPQA YVDGMAVGVK ELWQLLDISY DKFIRTTDDY           -       101 HEKVVAQVFE RLLAQDDIYL GEYSGWYSVS DEEFFTESQL AEVFRDEAGN            -       151 VTGGIAPSGH EVEWVSEESY FLRLSKYQDR LVEFFKAHPE FITPDGRLNE            -       201 MLRNFIEPGL EDLAVSRTTF TWGVPVPSNP KHVVYVWIDA LLNYATALGY            -       251 AQDEHGNFDK FWNGTVFHMV GKDILRFHSI YWPILLMMLD VKLPDRLIAH            -       301 GWFVMKDGKM SKSKGNVVYP EMLVERYGLD PLRYYLMRNL PVGSDGTFTP            -       351 EDYVGRINYE LANDLGNLLN RTVSMINKYF DGQIPAYVEG VTEFDHVLAE            -       401 VAEKSIADFH THMEAVDYPR ALEAVWTLIS RTNKYIDETA PWVLDKDEAL            -       451 RDQLASVMSH WQASIRVVAH LIEPFMMETS RAVLTQIGLE EVSSLENLSL            -       501 ADFPADVTVV AKGTPIFPRL NMEEEIAYIK EQMEGNKPAV EKEWNPDEVE            -       551 LKLNKDEIKF EDFDKVEIRV AEVKEVSKVE GSDKLLQFRL DAGDGEDRQI            -       601 LSGIAKYYPN EQELVGKKVQ IVANLKPRKM MKKYVSQGMI LSAEHDGKLT            -       651 LLTVDPAVPN GSVIG-(R.sub.2).sub.n -Y                               - (E) Sequences from Streptococcus pneumoniae metS polynucleotide ORF       sequence                                                                        [SEQ ID NO:3].                                                                5'-1 CTCAAACTAA ACAAGGATGA AATCAAGTTT GAAGACTTTG ACAAGGTTGA                    -   51 AATCCGTGTC GCAGAAGTCA AAGAAGTGTC TAAAGTAGAA GGTTCAGATA                 -  101 AGTTGCTTCA ATTCCGCTTG GATGCTGGTG ATGGAGAAGA TCGTCAGATT                 -  151 CTTTCAGGAA TTGCAAAATA CTATCCAAAT GAACAAGAAT TGGTCGGCAA                 -  201 GAAAGTTCAA ATCGTTGCTA ACCTCAAACC ACGTAAAATG ATGAAAAAAT                 -  251 ATGTCAGCCA GGGTATGATT CTCTCAGCTG AACATGATGG CAAATTAACC                 -  301 CTTCTCACAG TTGATCCAGC TGTACCAAAT GGAAGTGTGA TTGGGTAA                 3'                                                                              - (F) metS polypeptide sequence deduced from the polynucleotide ORF        sequence in this                                                                table [SEQ ID NO:4].                                                          NH.sub.2 -1 LKLNKDEIKF EDFDKVEIRV AEVKEVSKVE GSDKLLQFRL DAGDGEDRQI            -    51 LSGIAKYYPN EQELVGKKVQ IVANLKPRKM MKKYVSQGMI LSAEHDGKLT                -   101 LLTVDPAVPN GSVIG-COOH                                               __________________________________________________________________________

Deposited materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on 11 Apr. 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit On 17 Apr.1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli wassimilarly deposited with the NCIMB and assigned deposit number 40800.

The Streptococcus pneumoniae strain deposit is referred to herein as"the deposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length metS gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. § 112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1[SEQ ID NO: 2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of metS, and also those which have at least 70% identity to apolypeptide of Table 1 [SEQ ID NOS: 2 and 4] or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 [SEQ IDNOS: 2 and 4], and more preferably at least 90% similarity (morepreferably at least 90% identity) to a polypeptide of Table 1 [SEQ IDNOS: 2 and 4] and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to a polypeptide of Table 1 [SEQID NOS: 2 and 4] and also include portions of such polypeptides withsuch portion of the polypeptide generally containing at least 30 aminoacids and more preferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable I (D) [SEQ ID NO: 2] wherein, at the amino terminus, X ishydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, R₁ andR₂ is any amino acid residue, and n is an integer between 1 and 1000.Any stretch of amino acid residues denoted by either R group, where R isgreater than 1, may be either a heteropolymer or a homopolymer,preferably a heteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with metS polypeptides fragments maybe "free-standing," or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NOS: 2 and 4], orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Streptococcus pneumoniae,are also preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of metS, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause disease in anindividual, particularly a human

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the metS polypeptide havinga deduced amino acid sequence of Table 1 [SEQ ID NOS: 2 and 4] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NOS: 1 and 3], a polynucleotide of theinvention encoding metS polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Streptococcus pneumoniae0100993 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 [SEQ ID NOS: 1 and 3],typically a library of clones of chromosomal DNA of Streptococcuspneumoniae 0100993 in E. coli or some other suitable host is probed witha radiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 [SEQ ID NO: 1] was discovered in a DNA library derivedfrom Streptococcus pneumoniae 0100993.

The DNA sequence set out in Table 1 [ SEQ ID NOS: 1] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NOS: 2] with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art The start codon of the DNA in Table 1 isnucleotide number 1 and last codon that encodes an amino acid is number1995, the stop codon being the next codon following this last codonencoding an amino acid.

metS of the invention is structurally related to other proteins of themethionyl tRNA synthetase family, as shown by the results of sequencingthe DNA encoding metS of the deposited strain. The protein exhibitsgreatest homology to Bacillus stearothermophilus methionyl tRNAsynthetase protein among known proteins. metS of Table 1 [SEQ ID NO: 2]has about 58% identity over its entire length and about 76% similarityover its entire length with the amino acid sequence of Bacillusstearothermophilus methionyl tRNA synthetase polypeptide.

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 [SEQ ID NO: 1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro-protein sequence. The polynucleotidemay also contain non-coding sequences, including for example, but notlimited to non-coding 5' and 3' sequences, such as the transcribed,non-translated sequences, termination signals, ribosome binding sites,sequences that stabilize MRNA, introns, polyadenylation signals, andadditional coding sequence which encode additional amino acids. Forexample, a marker sequence that facilitates purification of the fusedpolypeptide can be encoded. In certain embodiments of the invention, themarker sequence is a hexa-histidine peptide, as provided in the pQEvector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad.Sci, USA 86: 821-824 (1989), or an HA tag (Wilson et al., Cell 37: 767(1984). Polynucleotides of the invention also include, but are notlimited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is the polynucleotide ofcomprising nucleotide 1 to 1995 set forth in SEQ ID NO: 1 of Table 1which encodes the metS polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C)[SEQ ID NO: 1] wherein, at the 5' end of the molecule, X ishydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal,R₁ and R₂ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae metShaving the amino acid sequence set out in Table 1 [SEQ ID NO: 2]. Theterm also encompasses polynucleotides that include a single continuousregion or discontinuous regions encoding the polypeptide (for example,interrupted by integrated phage or an insertion sequence or editing)together with additional regions, that also may contain coding and/ornon-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO: 2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingmetS variants, that have the amino acid sequence of metS polypeptide ofTable 1 [SEQ ID NO: 2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, that do not alter the properties and activitiesof metS.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding metS polypeptide having an amino acid sequence set out in Table1 [SEQ ID NOS: 2 and 4], and polynucleotides that are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding metS polypeptide ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 [SEQ ID NO: 1].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO: 1 or SEQ ID NO: 3 under stringent hybridization conditionswith a probe having the sequence of said polynucleotide sequence setforth in SEQ ID NO: 1 or a fragment thereof; and isolating said DNAsequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding metS and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the metS gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the metS gene may be isolated byscreening using the DNA sequence provided in SEQ ID NO: 1 to synthesizean oligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or MRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS: 1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the metS polynucleotides ofthe invention for use as diagnostic reagents. Detection of metS in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also"individual(s)"), particularly mammals, and especially humans, infectedwith an organism comprising the metS gene may be detected at the nucleicacid level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled metS polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g., Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding metS can be used to identify and analyze mutations. Examples ofrepresentative primers are shown below in Table 2.

                  TABLE 2                                                         ______________________________________                                        Primers for amplification of metS polynucleotides                                 SEQ ID NO   PRIMER SEOUENCE                                               ______________________________________                                        5           5'-ATGTCTGAAAAAAATTTTTATATT-3'                                      6             5'-TTACCCAATCACACTTCCATTTGG-3'                                ______________________________________                                    

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying metS DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, and most preferably otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, comprising determining from asample derived from an individual an increased level of expression ofpolynucleotide having the sequence of Table 1 [SEQ ID NO: 1]. Increasedor decreased expression of metS polynucleotide can be measured using anyone of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, amplification, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over- expression of metS protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a metSprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chainsimianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-metS or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against metS- polypeptide may be employedto treat infections, particularly bacterial infections and especiallyotitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenicaUy or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarily determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody , for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Humn. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS, 1986:83,9551), encapsulation of DNA in various forms ofliposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS 1984:81,5849).

Antagonists and agonists--assays and molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of metSpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising metS polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a metS agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the metS polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, ie., without inducing the effects of metS polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to calorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in metS polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for metS antagonists is a competitive assaythat combines metS and a potential antagonist with metS-bindingmolecules, recombinant metS binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. metS can be labeled, such as byradioactivity or a calorimetric compound, such that the number of metSmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing metS-induced activities, thereby preventing the action of metSby excluding metS from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of metS.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective MRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock metS protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial metS proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with metS, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Streptococcus pneumoniae infection. Also providedare methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector to direct expressionof metS, or a fragment or a variant thereof, for expressing metS, or afragment or a variant thereof in vivo in order to induce animmunological response, such as, to produce antibody and/or T cellimmune response, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether thatdisease is already established within the individual or not. One way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a metS or protein coded therefrom,wherein the composition comprises a recombinant metS or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid metS or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

A metS polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non- aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain metSprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation. For administration to mammals, and particularly humans, itis expected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO: 1 wasobtained from a library of clones of chromosomal DNA of Streptococcuspneumoniae in E. coli. The sequencing data from two or more clonescontaining overlapping Streptococcus pneumoniae DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO: 1. Libraries may beprepared by routine methods, for example: Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bsh1235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2

metS Characterization

The enzyme mediated incorporation of radiolabelled amino acid into tRNAmay be measured by the aminoacylation method which measures aminoacid-tRNA as trichloroacetic acid-precipitable radioactivity fromradiolabelled amino acid in the presence of tRNA and ATP (Hughes J,Mellows G and Soughton S, 1980, FEBS Letters, 122:322-324). Thusinhibitors of methionyl tRNA synthetase can be detected by a reductionin the trichloroacetic acid precipitable radioactivity relative to thecontrol. Alternatively the tRNA synthetase catalysed partial PPi/ATPexchange reaction which measures the formation of radiolabelled ATP fromPPi can be used to detect methionyl tRNA synthetase inhibitors (CalenderR & Berg P, 1966, Biochemistry, 5, 1681-1690).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 6                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1998 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  ATGTCTGAAA AAAATTTTTA TATTACGACG CCGATTTACT ATCCATCTGG - #GAAACTTC    AT    60                                                                        - -  ATCGGTTCTG CCTACACAAC TATCGCATGT GATGTCCTAG CACGTTACAA - #ACGCCTGAT    G   120                                                                         - -  GGNTACGATG TATTTTATCT GACAGGTCTT GATGAACATG GTCAGAAAAT - #CCAGCAGAA    A   180                                                                         - -  GCGGAAGAAG CTGGTATTAC ACCTCAAGCC TATGTTGATG GAATGGCGGT - #TGGAGTTAA    A   240                                                                         - -  GAACTCTGGC AATTACTAGA TATCTCATAC GATAAATTTA TCCGTACAAC - #CGATGACTA    C   300                                                                         - -  CATGAAAAAG TTGTCGCACA GGTCTTTGAA CGCTTACTTG CTCAAGATGA - #TATCTACTT    G   360                                                                         - -  GGTGAATACT CTGGTTGGTA TTCAGTATCA GACGAAGAAT TCTTTACAGA - #AAGCCAGCT    G   420                                                                         - -  GCAGAAGTTT TCCGTGATGA AGCTGGAAAT GTGACTGGTG GTATTGCTCC - #ATCAGGTCA    T   480                                                                         - -  GAGGTTGAAT GGGTTTCTGA AGAATCATAC TTCCTTCGCC TTAGCAAATA - #CCAAGACCG    T   540                                                                         - -  TTGGTCGAAT TTTTCAAAGC TCATCCTGAA TTTATCACGC CAGATGGTCG - #TCTTAATGA    A   600                                                                         - -  ATGCTACGCA ACTTCATCGA GCCAGGTTTG GAAGATTTGG CGGTATCTCG - #TACAACCTT    T   660                                                                         - -  ACATGGGGAG TGCCTGTCCC ATCAAATCCA AAACACGTTG TCTACGTTTG - #GATTGATGC    C   720                                                                         - -  CTTCTTAACT ATGCGACAGC TCTTGGCTAC GCTCAAGACG AACATGGTAA - #CTTTGACAA    G   780                                                                         - -  TTCTGGAATG GAACAGTCTT CCATATGGTA GGAAAAGACA TCCTTCGCTT - #CCACTCTAT    C   840                                                                         - -  TACTGGCCAA TCCTTCTTAT GATGTTGGAT GTTAAATTAC CTGATCGTTT - #GATTGCCCA    T   900                                                                         - -  GGTTGGTTTG TCATGAAAGA CGGAAAAATG TCTAAGTCAA AAGGGAATGT - #CGTTTACCC    T   960                                                                         - -  GAAATGTTGG TAGAGCGTTA TGGACTAGAT CCACTTCGTT ACTACCTCAT - #GCGTAACCT    T  1020                                                                         - -  CCAGTTGGTT CAGACGGAAC CTTTACTCCT GAAGACTATG TCGGTCGTAT - #CAACTATGA    A  1080                                                                         - -  TTGGCTAATG ACCTTGGGAA CCTCCTTAAC CGTACGGTTT CCATGATTAA - #TAAGTACTT    T  1140                                                                         - -  GATGGACAAA TCCCTGCCTA TGTAGAAGGT GTGACTGAAT TTGATCATGT - #TCTTGCTGA    G  1200                                                                         - -  GTTGCAGAAA AATCAATCGC AGACTTCCAT ACACACATGG AAGCAGTTGA - #TTATCCACG    T  1260                                                                         - -  GCGCTTGAAG CAGTCTGGAC TCTGATCTCT CGTACCAATA AATACATCGA - #TGAGACTGC    A  1320                                                                         - -  CCATGGGTCT TGGACAAGGA TGAAGCTCTT CGTGACCAAT TGGCAAGTGT - #CATGAGCCA    T  1380                                                                         - -  TGGCAAGCCA GCATTCGTGT AGTTGCTCAC TTGATTGAAC CATTTATGAT - #GGAAACTAG    T  1440                                                                         - -  CGTGCAGTTT TGACTCAAAT TGGTTTGGAA GAAGTTTCTA GTCTTGAAAA - #CTTGAGTTT    G  1500                                                                         - -  GCTGACTTCC CAGCAGATGT GACTGTAGTT GCCAAAGGAA CACCTATCTT - #TCCACGTCT    A  1560                                                                         - -  AATATGGAAG AAGAAATCGC CTATATCAAG GAACAAATGG AAGGCAATAA - #ACCAGCAGT    C  1620                                                                         - -  GAAAAAGAAT GGAATCCGGA CGAAGTTGAG CTCAAACTAA ACAAGGATGA - #AATCAAGTT    T  1680                                                                         - -  GAAGACTTTG ACAAGGTTGA AATCCGTGTC GCAGAAGTCA AAGAAGTGTC - #TAAAGTAGA    A  1740                                                                         - -  GGTTCAGATA AGTTGCTTCA ATTCCGCTTG GATGCTGGTG ATGGAGAAGA - #TCGTCAGAT    T  1800                                                                         - -  CTTTCAGGAA TTGCAAAATA CTATCCAAAT GAACAAGAAT TGGTCGGCAA - #GAAAGTTCA    A  1860                                                                         - -  ATCGTTGCTA ACCTCAAACC ACGTAAAATG ATGAAAAAAT ATGTCAGCCA - #GGGTATGAT    T  1920                                                                         - -  CTCTCAGCTG AACATGATGG CAAATTAACC CTTCTCACAG TTGATCCAGC - #TGTACCAAA    T  1980                                                                         - -  GGAAGTGTGA TTGGGTAA            - #                  - #                      - #1998                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 665 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  Met Ser Glu Lys Asn Phe Tyr Ile Thr Thr - #Pro Ile Tyr Tyr Pro Ser        1               5 - #                 10 - #                 15              - -  Gly Lys Leu His Ile Gly Ser Ala Tyr Thr - #Thr Ile Ala Cys Asp Val                   20     - #             25     - #             30                  - -  Leu Ala Arg Tyr Lys Arg Leu Met Gly Tyr - #Asp Val Phe Tyr Leu Thr               35         - #         40         - #         45                      - -  Gly Leu Asp Glu His Gly Gln Lys Ile Gln - #Gln Lys Ala Glu Glu Ala           50             - #     55             - #     60                          - -  Gly Ile Thr Pro Gln Ala Tyr Val Asp Gly - #Met Ala Val Gly Val Lys       65                 - # 70                 - # 75                 - # 80       - -  Glu Leu Trp Gln Leu Leu Asp Ile Ser Tyr - #Asp Lys Phe Ile Arg Thr                       85 - #                 90 - #                 95              - -  Thr Asp Asp Tyr His Glu Lys Val Val Ala - #Gln Val Phe Glu Arg Leu                   100     - #            105     - #            110                 - -  Leu Ala Gln Asp Asp Ile Tyr Leu Gly Glu - #Tyr Ser Gly Trp Tyr Ser               115         - #        120         - #        125                     - -  Val Ser Asp Glu Glu Phe Phe Thr Glu Ser - #Gln Leu Ala Glu Val Phe           130             - #    135             - #    140                         - -  Arg Asp Glu Ala Gly Asn Val Thr Gly Gly - #Ile Ala Pro Ser Gly His       145                 - #150                 - #155                 -         #160                                                                             - -  Glu Val Glu Trp Val Ser Glu Glu Ser Tyr - #Phe Leu Arg Leu Ser        Lys                                                                                              165 - #                170 - #                175            - -  Tyr Gln Asp Arg Leu Val Glu Phe Phe Lys - #Ala His Pro Glu Phe Ile                   180     - #            185     - #            190                 - -  Thr Pro Asp Gly Arg Leu Asn Glu Met Leu - #Arg Asn Phe Ile Glu Pro               195         - #        200         - #        205                     - -  Gly Leu Glu Asp Leu Ala Val Ser Arg Thr - #Thr Phe Thr Trp Gly Val           210             - #    215             - #    220                         - -  Pro Val Pro Ser Asn Pro Lys His Val Val - #Tyr Val Trp Ile Asp Ala       225                 - #230                 - #235                 -         #240                                                                             - -  Leu Leu Asn Tyr Ala Thr Ala Leu Gly Tyr - #Ala Gln Asp Glu His        Gly                                                                                              245 - #                250 - #                255            - -  Asn Phe Asp Lys Phe Trp Asn Gly Thr Val - #Phe His Met Val Gly Lys                   260     - #            265     - #            270                 - -  Asp Ile Leu Arg Phe His Ser Ile Tyr Trp - #Pro Ile Leu Leu Met Met               275         - #        280         - #        285                     - -  Leu Asp Val Lys Leu Pro Asp Arg Leu Ile - #Ala His Gly Trp Phe Val           290             - #    295             - #    300                         - -  Met Lys Asp Gly Lys Met Ser Lys Ser Lys - #Gly Asn Val Val Tyr Pro       305                 - #310                 - #315                 -         #320                                                                             - -  Glu Met Leu Val Glu Arg Tyr Gly Leu Asp - #Pro Leu Arg Tyr Tyr        Leu                                                                                              325 - #                330 - #                335            - -  Met Arg Asn Leu Pro Val Gly Ser Asp Gly - #Thr Phe Thr Pro Glu Asp                   340     - #            345     - #            350                 - -  Tyr Val Gly Arg Ile Asn Tyr Glu Leu Ala - #Asn Asp Leu Gly Asn Leu               355         - #        360         - #        365                     - -  Leu Asn Arg Thr Val Ser Met Ile Asn Lys - #Tyr Phe Asp Gly Gln Ile           370             - #    375             - #    380                         - -  Pro Ala Tyr Val Glu Gly Val Thr Glu Phe - #Asp His Val Leu Ala Glu       385                 - #390                 - #395                 -         #400                                                                             - -  Val Ala Glu Lys Ser Ile Ala Asp Phe His - #Thr His Met Glu Ala        Val                                                                                              405 - #                410 - #                415            - -  Asp Tyr Pro Arg Ala Leu Glu Ala Val Trp - #Thr Leu Ile Ser Arg Thr                   420     - #            425     - #            430                 - -  Asn Lys Tyr Ile Asp Glu Thr Ala Pro Trp - #Val Leu Asp Lys Asp Glu               435         - #        440         - #        445                     - -  Ala Leu Arg Asp Gln Leu Ala Ser Val Met - #Ser His Trp Gln Ala Ser           450             - #    455             - #    460                         - -  Ile Arg Val Val Ala His Leu Ile Glu Pro - #Phe Met Met Glu Thr Ser       465                 - #470                 - #475                 -         #480                                                                             - -  Arg Ala Val Leu Thr Gln Ile Gly Leu Glu - #Glu Val Ser Ser Leu        Glu                                                                                              485 - #                490 - #                495            - -  Asn Leu Ser Leu Ala Asp Phe Pro Ala Asp - #Val Thr Val Val Ala Lys                   500     - #            505     - #            510                 - -  Gly Thr Pro Ile Phe Pro Arg Leu Asn Met - #Glu Glu Glu Ile Ala Tyr               515         - #        520         - #        525                     - -  Ile Lys Glu Gln Met Glu Gly Asn Lys Pro - #Ala Val Glu Lys Glu Trp           530             - #    535             - #    540                         - -  Asn Pro Asp Glu Val Glu Leu Lys Leu Asn - #Lys Asp Glu Ile Lys Phe       545                 - #550                 - #555                 -         #560                                                                             - -  Glu Asp Phe Asp Lys Val Glu Ile Arg Val - #Ala Glu Val Lys Glu        Val                                                                                              565 - #                570 - #                575            - -  Ser Lys Val Glu Gly Ser Asp Lys Leu Leu - #Gln Phe Arg Leu Asp Ala                   580     - #            585     - #            590                 - -  Gly Asp Gly Glu Asp Arg Gln Ile Leu Ser - #Gly Ile Ala Lys Tyr Tyr               595         - #        600         - #        605                     - -  Pro Asn Glu Gln Glu Leu Val Gly Lys Lys - #Val Gln Ile Val Ala Asn           610             - #    615             - #    620                         - -  Leu Lys Pro Arg Lys Met Met Lys Lys Tyr - #Val Ser Gln Gly Met Ile       625                 - #630                 - #635                 -         #640                                                                             - -  Leu Ser Ala Glu His Asp Gly Lys Leu Thr - #Leu Leu Thr Val Asp        Pro                                                                                              645 - #                650 - #                655            - -  Ala Val Pro Asn Gly Ser Val Ile Gly                                                  660     - #            665                                        - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 348 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  CTCAAACTAA ACAAGGATGA AATCAAGTTT GAAGACTTTG ACAAGGTTGA - #AATCCGTGT    C    60                                                                         - -  GCAGAAGTCA AAGAAGTGTC TAAAGTAGAA GGTTCAGATA AGTTGCTTCA - #ATTCCGCTT    G   120                                                                         - -  GATGCTGGTG ATGGAGAAGA TCGTCAGATT CTTTCAGGAA TTGCAAAATA - #CTATCCAAA    T   180                                                                         - -  GAACAAGAAT TGGTCGGCAA GAAAGTTCAA ATCGTTGCTA ACCTCAAACC - #ACGTAAAAT    G   240                                                                         - -  ATGAAAAAAT ATGTCAGCCA GGGTATGATT CTCTCAGCTG AACATGATGG - #CAAATTAAC    C   300                                                                         - -  CTTCTCACAG TTGATCCAGC TGTACCAAAT GGAAGTGTGA TTGGGTAA - #                   348                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 115 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  Leu Lys Leu Asn Lys Asp Glu Ile Lys Phe - #Glu Asp Phe Asp Lys Val        1               5 - #                 10 - #                 15              - -  Glu Ile Arg Val Ala Glu Val Lys Glu Val - #Ser Lys Val Glu Gly Ser                   20     - #             25     - #             30                  - -  Asp Lys Leu Leu Gln Phe Arg Leu Asp Ala - #Gly Asp Gly Glu Asp Arg               35         - #         40         - #         45                      - -  Gln Ile Leu Ser Gly Ile Ala Lys Tyr Tyr - #Pro Asn Glu Gln Glu Leu           50             - #     55             - #     60                          - -  Val Gly Lys Lys Val Gln Ile Val Ala Asn - #Leu Lys Pro Arg Lys Met       65                 - # 70                 - # 75                 - # 80       - -  Met Lys Lys Tyr Val Ser Gln Gly Met Ile - #Leu Ser Ala Glu His Asp                       85 - #                 90 - #                 95              - -  Gly Lys Leu Thr Leu Leu Thr Val Asp Pro - #Ala Val Pro Asn Gly Ser                   100     - #            105     - #            110                 - -  Val Ile Gly                                                                      115                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  ATGTCTGAAA AAAATTTTTA TATT         - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -  TTACCCAATC ACACTTCCAT TTGG         - #                  - #                    24                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment encoding SEQID NO:
 2. 2. An isolated nucleic acid segment comprising a nucleotidesequence which is fully complementary to the polynucleotide of claim 1.3. An isolated vector comprising the polynucleotide segment of claim 1.4. An isolated vector comprising the nucleic acid segment of claim
 2. 5.An isolated host cell comprising the vector of claim
 3. 6. An isolatedhost cell comprising the vector of claim
 4. 7. A process for producing apolypeptide encoded by said polynucleotide segment comprising culturingthe host cell of claim 5 under conditions sufficient for the productionof said polypeptide.
 8. An isolated polynucleotide segment encoding amature polypeptide expressed by a polynucleotide comprising SEQ ID NO: 1in deposited strain NCIMB 40794 or NCIMB
 40800. 9. An isolated nucleicacid segment comprising a nucleotide sequence which is fullycomplementary to the polynucleotide segment of claim
 8. 10. An isolatedvector comprising the polynucleotide segment of claim
 8. 11. An isolatedvector comprising the nucleic acid segment of claim
 9. 12. An isolatedhost cell comprising the vector of claim
 10. 13. An isolated host cellcomprising the vector of claim
 11. 14. A process for producing themature polypeptide comprising culturing the host cell of claim 12 underconditions sufficient for the production of said polypeptide.